Lipid-encapsulated dual-cleaving endonuclease for dna gene editing

ABSTRACT

Methods to edit genes by administering a chimeric nuclease to a cell or organism without the use of a viral vector.

CROSS-REFERENCE

This application is a continuation of U.S. application Ser. No.17/514,484, filed Oct. 29, 2021, which is a continuation ofInternational application number PCT/IB2020/054229, filed May 4, 2020,which claims benefit of U.S. provisional application No. 62/842,586,filed May 3, 2019, and 63/019,423, filed May 3, 2020, each of which isherein incorporated by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Sep. 17, 2018, isSequence_Listing_ST25.txt and is 77 KB in size.

BACKGROUND OF THE INVENTION

There are an estimated 5,000-10,000 monogenic diseases, defined asinherited conditions arising from mutations on a single gene. Thesediseases often manifest during childhood and lead to a variety ofconditions and sometimes premature death. It has been estimated thattogether they will affect about 6% of people at some point in theirlives. Diagnosis and treatment for these diseases remain largelyinsufficient, and the care is primarily palliative, focusing on diseasemanagement without addressing the underlying genetic defects. There arealso many more diseases in which a mutation to a gene contributes to thepathogenesis of the disease.

Gene editing is a gene therapy approach that relies on designernucleases to recognize and cut specific DNA sequences, and subsequentlyexploits innate cellular DNA repair pathways, namely nonhomologous endjoining (NHEJ) and homology directed repair (HDR), to introduce targetedmodifications in the genome. Four nuclease families have been used inthis context: meganucleases, zinc-finger nucleases (ZFNs), transcriptionactivator-like effector nucleases (TALENs), and clustered regulatoryinterspaced short palindromic repeats associated RNA-guided Cas9(CRISPR-Cas9) nucleases. These can be designed to precisely introduce adouble stranded break at the target locus of interest. Gene editingopens up the possibility of permanently modifying a genomic sequence ofinterest by enabling targeted disruption, insertion, excision, andcorrection in both ex vivo and in vivo settings. While these advancesare expected to revolutionize the field at large, current gene-editingapproaches are limited by efficacy of modification, safety concernsrelated to the specificity of nucleases, and delivery of gene-editingtools to target cell types.

A component of the type II CRISPR system that constitutes the innateimmune system of bacteria, the Cas9 (CRISPR-associated) protein hascaused a paradigm shift in the field of genome editing due to itsease-of-use. Programming Cas9 to cleave a desired sequence is a simplematter of changing the sequence of the Cas9-associated guide RNA to becomplementary to the target site. The ease of programming Cas9 targetingcontrasts with the more intensive protein engineering that is requiredfor other reagents (zinc finger nucleases (ZFNs), meganucleases,transcription activator-like effector nucleases (TALENs)). Cas9, alongwith proteins from type III CRISPR systems have been used for a myriadof genome-editing applications in a diverse range of organisms and arenow entering the realm of therapeutic applications in humans.

Cystic fibrosis (CF) is an autosomal-recessive disease resulting frommutations in the CFTR gene, which encodes an epithelial anion channel.The CFTR protein, cystic fibrosis transmembrane conductance regulator,is found across a wide range of organs including pancreas, kidney,liver, lungs, gastrointestinal tracts, and reproductive tracts, makingCF a multiorgan disease. Mutations in CFTR lead to suboptimal iontransport and fluid retention, causing the prominent clinicalmanifestations of abnormal thickening of the mucus in lungs andpancreatic insufficiency. In the lung, dysfunctional CFTR hindersmucociliary clearance, rendering the organ susceptible to bacterialinfections and inflammation, ultimately leading to airway occlusion,respiratory failure, and premature death. CF remains the most common andlethal genetic disease among the Caucasian population with70,000-100,000 sufferers estimated worldwide, highlighting a real needfor the development of better treatments.

One major challenge to the development of a therapeutic strategy for CFis the wide diversity of mutation types. Delta F508 (deletion ofphenylalanine at codon 508) mutation, with a prevalence of >80% in CFpatients, is by far the most common, but more than 1,990 deleteriousCFTR-mutations have been described. These mutations cause premature stopcodons, aberrant splicing, incorrect protein folding or trafficking tothe cell surface, and dysfunctional CFTRs with limited channel-openingcapacity. Pharmacological interventions have been targeted to several ofthese processes and while drug administration is therapeutic in somegating mutation types, the commonly occurring delta F508 still requiresa more effective treatment. Pharmaceutical advancement in the care ofCF, however, does not address mutations resulting from aberrant splicingor premature stop codons; it is in these instances gene editing couldprove most beneficial.

Similarly, in the Western population, approximately 15% of patients withnon-small cell lung cancer (NSCLC) harbor an activating mutation intheir tumor in the EGF receptor (EGFR) gene.

Existing gene editing technologies, such as CRISPR-Cas9 (and Cas9fusions), meganucleases, zinc finger proteins, type IIS restrictionendonucleases (FokI and FokI fusions) and TALENS are limited in theability to introduce gene deletions of a specific length or toaccurately repair a target gene in a sufficient number of cells to bemeaningful as a therapeutic agent for many genetic diseases. Moreover,for highly programmable RNA-guided nucleases, such as the monomericCas9, studies suggest that the specificity for predictably binding,cleaving and repairing only their target sites is limited, raisingconcerns over potential deleterious changes to a cell's genomic DNA thatmay inadvertently cause a secondary disease in a patient. Last, mostnucleases are delivered in viral vectors. Viral vectors have thepotential for: existing immunity in many populations; immunogenicityafter treatment; and genotoxicity. No non-viral delivery method existstoday to safely deliver the nuclease to target cells and allow forcontrolled dosing of the nuclease in vivo.

There is an unmet need for improvements to said existing gene editingtechnologies to address the above concerns to make gene editingtechnologies more efficient and effective.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the lipid-encapsulateddual-cleaving nuclease (TevCas9) after it has been prepared [Componentsare not to scale]. The I-TevI Domain 10 is joined to the RNA-guidedNuclease (Cas9) Domain 12 via a Linker Domain 11. In the preferredembodiment, the formed particles also contain Guide RNA 13 and Donor DNA14. The aforementioned nuclease is contained in a Lipid Particle 15which has been shaped into a sphere using an extrusion process.

FIG. 2A to 2E is a diagram of the mechanism by which lipid-encapsulatedTevCas9 is internalized into a cell and nucleus to reach its target DNA.As illustrated in FIG. 2A, a cell 20 or cells 20 are exposed to thenovel lipid-encapsulated nuclease particles 21 containing the TevCas9 25either by in vivo or ex vivo administration. As shown in FIG. 2B, thelipid-encapsulated nuclease particle 21 is endocytosed into the cell 20.The endosome 22 goes through a maturation process in the cytosol and istargeted for degradation (FIG. 2C). On certain occasions, the TevCas9 25can escape the endosome 22 and enter the cytosol (FIG. 2D). Ineukaryotic organisms, the nuclease (TevCas9) 25 is targeted to thenucleus 23 of the cell 20 through one or more nuclear-localizationsequences (“NLS”). As depicted in FIG. 2E, through its nuclearlocalization sequence, TevCas9 25 can enter the nucleus 23 and when inthe nucleus 23, the TevCas9 nuclease 25 binds to and cleaves 26 thetarget genomic DNA 24 sequence.

FIG. 3A to 3E is a diagram of the mechanisms by which lipid-encapsulatedTevCas9 modifies target DNA. The I-TevI domain 27 targets the I-TevITarget Sequence 29. The linker domain 30 joins the I-TevI domain 27 withthe Cas9 domain 28 which targets the Cas9 Target Sequence 31. The genemutation 32 is surrounded by or in close proximity to the I-TevI TargetSequence 29 and the Cas9 Target Sequence 31. As shown in FIG. 3B, theTevCas9 25 cleaves the target sequence leaving a deletion product 34 ofa predictable size with non-complementary DNA ends 35, 36. FIG. 3Cillustrates that in the presence of single-stranded donor DNA withhomology arms 37, the cell 20 can insert the donor DNA 37 sequence nearthe cut sites through the homology-directed repair (HDR) pathway 38.FIG. 3D illustrates that in the presence of donor DNA 39 with compatibleDNA ends to those cleaved by TevCas9 25, the cell 20 can insert thedonor DNA sequence 39 between the cut sites through directed-ligationusing the non-homologous end joining (NHEJ) pathway 40. In the absenceof donor DNA, the cell 20 can join the DNA ends through the NHEJ pathway40 (FIG. 3E).

FIG. 4A evidences that TevCas9, targeted to the CFTR gene using guide inSEQ ID 15, cleaves CFTR DNA substrate in vitro. FIG. 4B are cellstransfected with a plasmid DNA version of TevCas9 fused to a cleavableGFP tag imaged using phase contrast and GFP imaging on a Cytation5(Biotek Instruments Inc, VT, USA) after 48 hours treatment. Genomic DNAis extracted from harvested cells and editing at the CFTR gene isdetected by PCR amplification and a T7 Endonuclease I cleavage assay.

FIG. 5 evidences that TevCas9, targeted to the CFTR Delta F508 mutationusing guide in SEQ ID 21, cleaves a DNA substrate containing the CFTRDelta F508 mutation, but not substrate containing the wild-type CFTRsequence in vitro.

FIG. 6A illustrates that the saCas9 D10E mutation slows the conversionof nicked supercoiled DNA to linear DNA. FIG. 6B evidences that onlinear EMX1 DNA substrate, saCas9D10E (D10E) ribonucleoprotein complex(RNP) cleaves the target substrate to a similar level as saCas9wild-type (WT). Levels of editing by SaCas9D10E at computationallypredicted off-targets is lower than levels of editing by wild-typesaCas9 at the same off-targets.

FIG. 7A is a schematic of spacing of the I-TevI sites in the EGFR Exon19 deletion and wild-type (WT) EGFR. FIGS. 7B and 7C evidences thatTevCas9 containing the nicking mutation in Cas9 (H557A) targeted to EGFRusing the guide RNA in SEQ ID 16 cleaves EGFR Exon 19 deletion DNAsubstrate at a 4-fold faster rate than wild type EGFR. FIG. 7D areimages of HCC827 cells harbouring the EGFR Exon 19 deletion mutationtreated with TevCas9 targeted to EGFR are selectively killed compared toNuLi-1 cells harbouring wild-type EGFR (WT).

BRIEF SUMMARY OF THE INVENTION

The instant invention is directed to a chimeric nuclease comprising amodified I-TevI nuclease domain, preferably deleting Met¹ and havingLys²⁶ (which is Lys²⁷ in the untruncated version of I-TevI) and/or Cys³⁹(which is Cys⁴¹ in the untruncated version of I-TevI) modification, alinker, in particular SEQ ID NOS: 7-12 or fragments thereof and/orcontaining one or more of the following mutations Thr⁹⁵ (as referencedto the full-length I-TevI), Val¹¹⁷, Lys¹³⁵, Gln¹⁵⁸ or Asn¹⁴⁰, and amodified RNA-guided nuclease Staphylacoccus aureus Cas9 that may be thewild-type or a modified version, preferably containing a Glu¹⁰ or anAla⁵⁵⁷ mutation thereof wherein said I-TevI polypeptide comprises theentire amino acid sequence of SEQ ID NO: 6 or a fragment thereof, andguide RNA, in particular, SEQ ID NOS: 15, 16 or 21 or fragments thereof,that targets the Cas9 domain and a pharmaceutically-acceptableformulation comprising the chimeric nuclease, cationic and/or neutrallipid nanoparticles, optionally DNA-binding compounds, in particularGL67 (N⁴-cholesteryl-spermine) and a pharmaceutically acceptable carrierthereof.

In a further embodiment of the instant invention, in the formulation thelipid nanoparticle may contain exogenous donor DNA.

Another embodiment of the invention is directed to methods to edit genesby administering a chimeric nuclease to a cell or organism without theuse of a viral vector by using a controlled dose in vivo.

Another embodiment of the invention is directed to methods to deletedefined lengths of a DNA molecule or to replace select sequences from aDNA molecule by delivering a chimeric nuclease in vivo to a wholeorganism or to isolated cells in culture ex vivo wherein said cells aremammalian cells, bacteria, insect cells or plant cells.

In yet another embodiment, the novel chimeric nuclease targets twoindependent target sites on a select DNA molecule either cleaving at onetarget site or at both target sites and creating fragments that are 30to 36 nucleotides in length.

In a further example, the novel, purified chimeric nuclease furthercomprises a guide RNA.

Another aspect of the instant inventions is the use of an extrusionprocess creating particles of approximately 100 nM in diametercomprising an excipient wherein the excipient is selected from the groupconsisting of polysorbates, polyphosphates, calcium chlorides, sodiumchloride, sodium citrates, sodium hydroxide, sodium phosphates, sodiumethylenediaminetetraacetic acid, potassium chloride, potassium phosphateand starches, or mixtures of these substances so that the novel chimericnuclease can be administered to a patient using a nebulizer containingsaid formulation.

In a preferred embodiment, the instant invention is directed to a methodof treating a lung-related disease in a patient in need thereof byadministering a novel chimeric nuclease that modifies the DNA of lungepithelial cells wherein the chimeric nuclease replaces the CFTR deltaF508 mutation from the CFTR gene in an effort to treat cystic fibrosisor cleaves an EGFR exon 19 deletion in an effort to treat non-small celllung cancer.

In yet another embodiment, the invention is directed to a chimericnuclease comprising a modified I-TevI nuclease domain, a linker and amodified RNA-guided nuclease Staphylacoccus aureus Cas9 wherein saidRNA-guided nuclease Staphylacoccus aureus Cas9 contains Ala¹⁰, Ala⁵⁵⁷ orAla⁵⁸⁰ mutations and targets the EGFR exon 19 deletions of the EGFRgene.

In a further embodiment, wherein said guide RNA targets a specific CTFRgene sequence to cleave out the CFTR delta F508 mutation or a specificEGFR gene sequence that contains an EGFR exon 19 deletion mutation.

The instant invention also covers linkers comprising SEQ TD NOS: 7-12 orfragments thereof and modified donor DNA molecules selected from thegroup consisting of a linear single-strand of DNA comprising homologousregions flanking the sites targeted and/or cleaved by a chimericnuclease, a linear double-strand DNA comprising homologous regionsflanking the sites targeted and/or cleaved by a chimeric nuclease, adouble strand DNA of the same length comprising complimentary DNA endsto those cleaved by a chimeric nuclease, a circular double-strand DNAcomprising homologous regions flanking the sites targeted and/or cleavedby a chimeric nuclease, and a circular double-strand DNA comprising anI-TevI target site and a Cas9 target site wherein the product cleavedfrom the double-strand DNA contains complimentary ends to the endscleaved by a chimeric nuclease.

In a further example consists of a chimeric nuclease comprising amodified GIY-YIG nuclease domain, a linker and a modified RNA-guidednuclease Staphylacoccus aureus Cas9 or Streptococcus pyogenes Cas9 orEQR Streptococcus pyogenes Cas9 variant containing a Glu¹⁰ mutation (SEQID NO:19) and/or a Ala⁸⁴¹ mutation and/or a mutation that cleaves thesugar phosphate backbone of a target DNA on one strand of a target DNAwherein said GIY-YIG nuclease domain is selected from the gene familyconsisting of I-Bmol and Eco29kI.

In yet a further embodiment, the instant invention includes a chimericnuclease comprising a modified I-TevI nuclease domain, a linker and amodified nuclease or DNA targeting domain wherein said modified nucleaseor DNA targeting domain is selected from the group consisting ofLAGLIDADG, His-Cys Box, H—N—H, PD-(D/E)×K and Vsr-like meganucleases,zinc-finger nuclease, CRISPR protein selected from the group consistingof scCas9 (Streptococcus canis), fnCas9 (Francisella novicida), cjCas9(Campylobacter jejuni), Cpf1 (Lachnospiraceae bacterium), Cas12a(Acidaminococcus Sp), Cas13a (Leptorichia shahii) and Cas3(Streptococcus thermophilus) and DNA binding domain selected from thegroup consisting of zinc-finger motifs and TALE activator domains.

In an even further example, the instant invention covers a modifiedRNA-guided nuclease Staphylacoccus aureus Cas9 and a guide RNA whereinsaid guide RNA contains sequences that target genetic polymorphisms,different sequences in the CFTR or EGFR genes, sequences that retarget anuclease, bridged nucleic acids and/or a mixture of guide RNAs.

DETAILED DESCRIPTION OF THE INVENTION Definitions and Acronyms

For convenience, certain terms employed in the specification, examplesand appended claims are collected here. These definitions should be readin light of the disclosure and understood as by a person of skill in theart. Unless defined otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood by a person ofordinary skill in the art.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. The term “and/or” as used herein is defined as the possibilityof having one or the other or both. For example, “A and/or B” providesfor the scenarios of having just A or just B or a combination of A andB. If the claim reads A and/or B and/or C, the composition may include Aalone, B alone, C alone, A and B but not C, B and C but not A, A and Cbut not B or all three A, B and C as components.

The term “bioavailable” is art-recognized and refers to a form of thesubject disclosure that allows for it, or a portion of the amountadministered, to be absorbed by, incorporated to, or otherwisephysiologically available to a subject or patient to whom it isadministered.

The term “exogenous donor DNA”, as used herein, refers to any sequenceof DNA that, in whole or in part, is not the same as the original targetDNA sequence.

The term “flexible linker”, as used herein, refers to a situation whenthe RNA-guided nuclease domain (Cas9) binds to the target DNA sequence,the amino acid linker domain ensures mobility of the I-TevI domain toallow for recognition, binding and cleaving of its target sequence undercell physiological conditions (typically: pH ˜7.2, temperature ˜37° C.,[K+] ˜140 mM, [Na+]˜5-15 mM, [Cl−] ˜4 mM, [Ca++] ˜0.0001 mM). The lengthof the amino acid linker can influence how many nucleotides arepreferred between the Cas9 target site and the I-TevI target site.Certain amino acids in the linker may also make specific contacts withthe DNA sequence targeted by TevCas9. These linker-DNA contacts canaffect the flexibility of the I-TevI domain. Substituting amino acids inthe linker domain may affect the ability of the linker domain to makecontact with DNA.

The term “including”, as used herein, is used to mean “including but notlimited to”. “Including” and “including but not limited to” are usedinterchangeably.

The terms “inhaled administration”, “inhale”, “inhaled”, “inhalation” or“inhalation therapy”, which may be used interchangeably and as usedherein, include administration of a substantially uniform distributionof appropriately sized particles to the respiratory epithelium of thenose, central airways, the peripheral aspect of the lung and/or thealveolar region of the lung or by intratracheal instillation. Suchparticles may be introduced to the patient and/or produced using anappropriate device, preferably a nebulizer.

The term “patient,” “subject” or “host” to be treated by the subjectmethod may mean either a human or non-human animal. Non-human animalsinclude companion animals (e.g. cats, dogs) and animals raised forconsumption (i.e. food animals), such as cows, pigs, and chickens.

The term “pharmaceutically acceptable carrier” is art-recognized andrefers to a pharmaceutically-acceptable material, composition orvehicle, such as a liquid or solid filler, diluent, excipient, solventor encapsulating material, involved in carrying or transporting anysubject composition or component thereof from one organ, or portion ofthe body, to another organ, or portion of the body. Each carrier must be“acceptable” in the sense of being compatible with the subjectcomposition and its components and not injurious to the patient. Someexamples of materials which may serve as pharmaceutically acceptablecarriers include: (1) sugars, such as dextrose, lactose, glucose andsucrose; (2) starches, such as corn starch and potato starch; (3)cellulose, and its derivatives, such as microcrystalline cellulose,sodium carboxymethyl cellulose, methyl cellulose, ethyl cellulose,hydroxypropylmethyl cellulose (HPMC), and cellulose acetate; (4)glycols, such as propylene glycol; (5) polyols, such as glycerin,sorbitol, mannitol and polyethylene glycol; (6) esters, such as ethyloleate, glyceryl behenate and ethyl laurate; (7) buffering agents, suchas monobasic and dibasic phosphates, Tris/Borate/EDTA andTris/Acetate/EDTA (8) pyrogen-free water; (9) isotonic saline; (10)Ringer's solution; (11) ethyl alcohol; (12) phosphate buffer solutions;(13) polysorbates; (14) polyphosphates; and (15) other non-toxiccompatible substances employed in pharmaceutical formulations. Thedisclosed excipients may serve more than one function. For example, asolubilizing agent may also be a suspension aid, an emulsifier, apreservative, and the like.

In certain preferred embodiments, the pharmaceutically acceptableexcipient is a crystalline bulking excipient. The terms “crystallinebulking excipient” or “crystalline bulking agent” as used herein meansan excipient which provides bulk and structure to the lyophilizationcake. These crystalline bulking agents are inert and do not react withthe protein or nucleic acid. In addition, the crystalline bulking agentsare capable of crystallizing under lyophilization conditions. Examplesof suitable crystalline bulking agents include hydrophilic excipients,such as, water soluble polymers; sugars, such as mannitol, sorbitol,xylitol, glucitol, ducitol, inositiol, arabinitol, arabitol, galactitol,iditol, allitol, maltitol, fructose, sorbose, glucose, xylose,trehalose, allose, dextrose, altrose, lactose, glucose, fructose,gulose, idose, galactose, talose, ribose, arabinose, xylose, lyxose,sucrose, maltose, lactose, lactulose, fucose, rhamnose, melezitose,maltotriose, raffinose, altritol, their optically active forms (D- orL-forms) as well as the corresponding racemates; inorganic salts, bothmineral and mineral organic, such as, calcium salts, such as thelactate, gluconate, glycerylphosphate, citrate, phosphate monobasic anddibasic, succinate, sulfate and tartrate, as well as the same salts ofaluminum and magnesium; carbohydrates, such as, the conventional mono-and di-saccharides as well as the corresponding polyhydric alcohols;proteins, such as, albumin; amino acids, such as glycine; emulsifiablefats and polyvinylpyrrolidone. Preferred crystalline bulking agents areselected from the group consisting of glycine, mannitol, dextran,dextrose, lactose, sucrose, polyvinylpyrrolidone, trehalose, glucose andcombinations thereof. Particularly useful bulking agents includedextran.

The term “pharmaceutically-acceptable salts”, as used herein, isart-recognized and refers to the relatively non-toxic, inorganic andorganic acid addition salts, or inorganic or organic base addition saltsof compounds, including, for example, those contained in compositions ofthe present invention. Some examples of pharmaceutically-acceptablesalts include: (1) calcium chlorides; (2) sodium chlorides; (3) sodiumcitrates; (4) sodium hydroxide; (5) sodium phosphates; (6) sodiumethylenediaminetetraacetic acid; (7) potassium chloride; (8) potassiumphosphate; and (9) other non-toxic compatible substances employed inpharmaceutical formulations.

The term “substitution”, as used herein, refers to the replacement of anamino acid in a sequence with a different amino acid. As used herein,the shorthand X10Y indicates that amino acid Y has been “substituted”for amino acid X found in the 10^(th) position of the sequence. As anexample, W26C denotes that amino acid Tryptophan-26 (Trp, W) is changedto a Cysteine (Cys). Similarly, the notation AA^(X) indicates that AA isan amino acid that replaced the amino acid found in the X position. Asan example, Lys²⁶ denotes the replacement of the amino acid in the26^(th) position in a sequence with Lysine. Use of either shorthand isinterchangeable. In addition, use of the one- or three-letterabbreviations for an amino acid is also interchangeable.

The term “therapeutic agent”, as used herein, is art-recognized andrefers to any chemical or biochemical moiety that is a biologically,physiologically, or pharmacologically active substance that acts locallyor systemically in a subject. Examples of therapeutic agents, alsoreferred to as “drugs”, are described in well-known literaturereferences such as the Merck Index, the Physician's Desk Reference, andThe Pharmacological Basis of Therapeutics, and they include, withoutlimitation, medicaments; vitamins; mineral supplements; substances usedfor the treatment, prevention, diagnosis, cure or mitigation of adisease or illness; substances which affect the structure or function ofthe body; or pro-drugs, which become biologically active or more activeafter they have been placed in a physiological environment.

The term “therapeutic effect”, as used herein, is art-recognized andrefers to a local or systemic effect in animals, particularly mammals,and more particularly humans caused by a pharmacologically activesubstance. The term thus means any substance intended for use in thediagnosis, cure, mitigation, treatment, or prevention of disease or inthe enhancement of desirable physical or mental development and/orconditions in an animal or human. The phrase “therapeutically-effectiveamount” means that amount of such a substance that produces some desiredlocal or systemic effect at a reasonable benefit/risk ratio applicableto any treatment. The therapeutically effective amount of such substancewill vary depending upon the subject and disease condition beingtreated, the weight and age of the subject, the severity of the diseasecondition, the manner of administration and the like, which can readilybe determined by one of ordinary skill in the art. For example, certaincompositions of the present invention may be administered in asufficient amount to produce a at a reasonable benefit/risk ratioapplicable to such treatment.

The term “treating”, as used herein, includes any effect, e.g.,lessening, reducing, modulating, or eliminating, that results in theimprovement of the condition, disease, disorder, and the like. As usedherein, “treating” can include both prophylactic, and therapeutictreatment. For example, therapeutic treatment can include delayinginhibiting or preventing the progression of cystic fibrosis or non-smallcell lung cancer, the reduction or elimination of symptoms associatedwith cystic fibrosis or non-small cell lung cancer. Prophylactictreatment can include preventing, inhibiting or delaying the onset ofcystic fibrosis or non-small cell lung cancer.

As used herein, an “effective amount” refers to an amount sufficient toelicit the desired response. In the present invention, the desiredbiological response is the treatment of cystic fibrosis and/or non-smallcell lung cancer (NSCLC).

A “buffer” as used herein is any acid or salt combination which ispharmaceutically acceptable and capable of maintaining the compositionof the present invention within a desired pH range. Buffers in thedisclosed compositions maintain the pH in a range of about 2 to about8.5, about 5.0 to about 8.0, about 6.0 to about 7.5, about 6.5 to about7.5, or about 6.5. Suitable buffers include, any pharmaceuticalacceptable buffer capable of maintaining the above pH ranges, such as,for example, acetate, tartrate phosphate or citrate buffers. In oneembodiment, the buffer is a phosphate buffer. In another embodiment thebuffer is an acetate buffer. In one embodiment the buffer is disodiumhydrogen phosphate, sodium chloride, potassium chloride and potassiumphosphate monobasic.

In the disclosed compositions the concentration of buffer is typicallyin the range of about 0.1 mM to about 1000 mM, about 0.2 mM to about 200mM, about 0.5 mM to about 50 mM, about 1 mM to about 10 mM or about 6.0mM.

As used herein, an “anti-microbial agent” is a pharmaceuticallyacceptable preservative, suitable for administration to a subject, whichinhibits, prevents, or delays the growth or microorganisms including,for example bacteria, viruses and fungi in the compositions of thepresent invention. Suitable anti-microbial agents for use in thecompositions and methods of the present invention include, but are notlimited to, cresols, benzyl alcohol, phenol, benzalkonium chloride,benzethonium chloride, chlorobutanol, phenylethyl alcohol, methylparaben, propyl paraben, thiomersal and phenylmercuric nitrate andacetate. In one embodiment the anti-microbial agents is m-cresol,chlorocresol or phenol. In another embodiment the anti-microbial agentsis chlorocresol or phenol. In another embodiment the anti-microbialagents is phenol.

As used herein an effective amount of an anti-microbial agent is anamount effective to inhibit, prevent or delay the growth ormicroorganisms including, for example bacteria, viruses, and fungi inthe compositions of the present invention. In the compositions of thepresent invention, the amount of anti-microbial agent is typically inthe range from about 0.1 to about 20 mg/ml, about 0.2 to about 30 mg/ml,about 0.2 to about 10 mg/ml, about 0.25 to about 5 mg/ml, about 0.5 toabout 50 mg/ml, about 1 to about 10 mg/ml, about 3 mg/ml or about 5mg/ml.

The compositions of the present invention can also be lyophilized usinglyophilization techniques known in the art and stored as a powder whichcan be reconstituted prior to administration. The term “lyophilization”as used herein is a freeze drying or dehydration technique whichinvolves removing a solvent, preferably a water miscible solvent, morepreferably water from a composition or the present invention, typicallyby sublimation under high vacuum when the composition is in a frozenstate. Typically, lyophilization is carried out in lyophilizationequipment (a lyophilizer), which comprises a drying chamber withvariable temperature controls, a condenser to collect water, and avacuum system to reduce the pressure in the drying chamber.

The terms “lyophilized composition”, as used herein mean the solidresidue or powder which is produced, or which remains after thelyophilization procedure as defined above. The lyophilized compositionof the present invention typically further comprises a pharmaceuticallyacceptable excipient. The term “pharmaceutically acceptable excipient”as used herein refers to a substance which is added to a solution priorto lyophilization to enhance characteristics such as the color, texture,strength, and volume of the lyophilized cake. Pharmaceuticallyacceptable excipients may be, for example, buffers and pH adjusters,crystalline bulking excipients, stabilizers, and tonicity raisingagents.

As used herein, a stabilizer is a composition which maintains thechemical, biological or stability of the chimeric nuclease. Examples ofstabilizing agent include polyols, which includes a saccharide,preferably a monosaccharide or disaccharide, e.g., glucose, trehalose,raffinose, or sucrose; a sugar alcohol such as, for example, mannitol,sorbitol or inositol, a polyhydric alcohol such as glycerin or propyleneglycol or mixtures thereof and albumin.

A pharmaceutically acceptable salt is a salt which is suitable foradministration to a subject, such as, a human. The chimeric nuclease ofthe present invention can have one or more sufficiently acidic protonthat can react with a suitable organic or inorganic base to form a baseaddition salt. Base addition salts include those derived from inorganicbases, such as ammonium or alkali or alkaline earth metal hydroxides,carbonates, bicarbonates, and the like, and organic bases such asalkoxides, alkyl amides, alkyl and aryl amines, and the like. Such basesuseful in preparing the salts of this invention thus include sodiumhydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate,and the like. The chimeric nuclease of the present invention having asufficiently basic group, such as an amine can react with an organic orinorganic acid to form an acid addition salt. Acids commonly employed toform acid addition salts from compounds with basic groups are inorganicacids such as hydrochloric acid, hydrobromic acid, hydroiodic acid,sulfuric acid, phosphoric acid, and the like, and organic acids such asp-toluenesulfonic acid, methanesulfonic acid, oxalic acid,p-bromophenyl-sulfonic acid, carbonic acid, succinic acid, citric acid,benzoic acid, acetic acid, and the like. Examples of such salts includethe sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate,monohydrogenphosphate, dihydrogenphosphate, metaphosphate,pyrophosphate, chloride, bromide, iodide, acetate, propionate,decanoate, caprylate, acrylate, formate, isobutyrate, caproate,heptanoate, propiolate, oxalate, malonate, succinate, suberate,sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate,benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate,hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate,phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate,gamma-hydroxybutyrate, glycolate, tartrate, methanesulfonate,propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate,mandelate, and the like.

While specific embodiments of the subject invention have been discussed,the above specification is illustrative and not restrictive. Manyvariations of the invention will become apparent to those skilled in theart upon review of this specification. The full scope of the inventionshould be determined by reference to the claims, along with their fullscope of equivalents, and the specification, along with such variations.

Unless otherwise indicated, all numbers expressing quantities ofingredients, reaction conditions, and so forth used in the specificationand claims are to be understood as being modified in all instances bythe term “about.” Accordingly, unless indicated to the contrary, thenumerical parameters set forth in this specification and attached claimsare approximations that may vary depending upon the desired propertiessought to be obtained by the present invention.

The above discussion is meant to be illustrative of the principle andvarious embodiments of the present invention. Numerous variations andmodifications will become apparent to those skilled in the art once theabove disclosure is fully appreciated. It is intended that the followingclaims be interpreted to embrace all such variations and modifications.

Abbreviations

Abbreviations used herein are defined as follows:

-   AA amino acid-   Cas9 CRISPR-associated protein 9-   CF Cystic fibrosis-   CFTR Cystic fibrosis transmembrane conductance regulator gene-   cjCas9 Campylobacter jejuni Cas9-   Cpf1 CRISPR from Prevotella and Francisella 1-   CRISPR Clustered Regulatory Interspaced Short Palindromic Repeats-   DLS Dynamic Light Scattering-   DMEM Dulbecco's Modified Eagle's Medium-   DMPE 1,2-ditetradecanoyl-sn-glycero-3-phosphoethanolamine-   DNA deoxyribonucleic acid-   DOAB dioctadecyldimethylammonium bromide-   DOPE 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine-   DPPC Dipalmitoylphosphatidylcholine-   E. Coli Escherichia coli-   EDTA ethylenediaminetetraacetic acid-   EGFR Epidermal growth factor receptor-   ELISA Enzyme-linked immunosorbent assay-   fnCas9 Francisella novicida Cas9-   HDR Homology directed repair-   IMAC Immobilized Metal Affinity Chromatography-   IPTG Isopropyl β-D-1-thiogalactopyranoside-   MPEG-5000-DMPE N-(carbonyl-methoxypolyethyleneglycol 5000)-1,2    dipalmitoyl-sn-glycero-3-phosphoethanolamine-   NSCLC Non-small cell lung cancer-   NHEJ Non-homologous end joining-   NLS Nuclear-localized signal-   PC Phosphatidylcholine-   PCR polymerase chain reaction-   PE Phosphoethanolamine-   RNA ribonucleic acid-   saCas9 Staphylococcus aureus Cas9-   scCas9 Streptococcus canis Cas9-   SDS sodium dodecyl sulfate-   spCas9 Streptococcus pyogenes Cas9-   TALEN Transcription activator-like effector nucleases-   TEV Tobacco Etch Virus-   TevCas9 Modified I-TevI domain, a linker peptide and modified    RNA-guided nuclease Staphylococcus aureus Cas9-   ZFN zinc-finger nucleases

The Inventors discovered a chimeric nuclease comprising a modifiedversion of an I-TevI domain, a linker peptide and a modified version ofan RNA-guided nuclease Staphylococcus aureus Cas9 (“saCas9”)(hereinafter referred to as “TevCas9”) that, when mixed with a lipidnanoparticle, with or without exogenous donor DNA, when delivered tocells, replaces DNA sequences in the presence of exogenous donor DNA ordeletes defined lengths of DNA in the absence of exogenous donor DNA.The novel chimeric nuclease has been shown to edit genes in human cells,but also cells of other organisms such as bacteria, yeast, insect,plant, or other mammals, either in whole organisms (in vivo) or inisolated cells cultures (ex vivo).

The novel chimeric nucleases discovered by the present Inventors presentthe following advantages over existing gene editing technologies andmethods, in particular,

-   -   a. The nuclease, which is a modified version of the TevCas9        nuclease, is capable of targeting two independent target sites        as a single protein and cleaving the DNA at one or both of these        sites. It can be reprogrammed to many different target DNA        sequences through modifying one or more of the I-TevI domain,        the linker domain, the Cas9 domain or the guide RNA (which        targets the Cas9 domain to its target sequence);    -   b. If the nuclease cleaves at two sites, it cleaves out precise        lengths of DNA (˜30-36 bases depending on the sites targeted by        I-TevI and Cas9);    -   c. The Cas9 domain contains a mutation (D10E) which is        rationally designed to modify the Cas9 nuclease activity and/or        increase the Cas9 domain's specificity for its target binding        site;    -   d. In the presence of exogenous donor DNA, the invention is        designed to replace target DNA sequences in a higher percentage        of cells than existing technologies or practices;    -   e. The nuclease can be purified as a single contiguous protein        combined with a guide RNA, which simplifies manufacturing;    -   f. The lipid nanoparticle allows for non-viral delivery to        target cells with high efficiency and low toxicity, allowing for        controlled dosing of the nuclease. Although other lipid-based        nuclease delivery technologies exist, none are of a composition        suitable for use in vivo;    -   g. The lipid nanoparticles are also designed for the delivery of        nuclease through nebulization (inhalation);    -   h. One version of the nuclease targets and cleaves the CFTR gene        to correct the CFTR delta F508 mutation for the treatment of        Cystic Fibrosis (SEQ ID NO 1); and    -   i. Another version of the nuclease is designed to target and        cleave the clinically relevant EGFR exon 19 deletion mutations        (SEQ ID NOS 2-4), which are present in a variety of cancers,        including non-small-cell lung cancer (NSCLC).

The fusion of a GIY-YIG nuclease, such as I-TevI, through a flexiblelinker to DNA binding domains is known (WO2014/121222). A prior versionof the dual-cleaving TevCas9 has been described which comprises aminoacids 1-92 of the wild-type I-TevI nuclease domain, a linker regioncomprising amino acids 93-169 of I-TevI linker region and theStreptococcus pyogenes Cas9 (“spCas9”)(Wolfs J M et al., (2016),‘Biasing Genome-Editing Events Toward Precise Length Deletions with anRNA-Guided TevCas9 Dual Nuclease,’ Proc Natl Acad Sci USA,113(52):14988-93). The chimeric nuclease of the invention comprises thefollowing:

-   -   i. An I-TevI nuclease domain which binds a new target sequence        allowing to target clinically relevant gene sequences, such as        the CFTR gene;    -   ii. Various flexible linker regions intended to confer different        DNA binding or nuclease activity to TevCas9;    -   iii. A saCas9 nuclease domain (US-1988/065406 B2). The use of        saCas9 over spCas9 results in a smaller DNA coding sequence        (˜3.7 kilobases for Tev-saCas9 versus ˜4.6 kilobases for        Tev-spCas9) and lower molecular weight TevCas9 protein (˜144        kilodaltons for Tev-saCas9 versus ˜179 kilodaltons for        Tev-spCas9) which is more amenable to multiple delivery        technologies; cleaving by the saCas9 domain between the 3^(rd)        and 4^(th) nucleotide is predictable compared to spCas9 which is        more amenable to defined length deletions, as discovered by the        inventors of the claimed technology.    -   iv. One version where the guide RNA is targeted to specific CFTR        gene sequence near the CFTR delta F508 mutation; and    -   v. A second version where the guide RNA is targeted to specific        EGFR gene sequences and is intended to cleave only DNA with        appropriated spaced I-TevI site and Cas9 target site. Such        appropriately spaced sites occur in certain EGFR exon 19        deletion mutations (SEQ ID NO 2-4) but not in wild-type EGFR        (SEQ ID NO 5);    -   a. The invention comprises lipid nanoparticles of certain        compositions that are selectively sized to a mean diameter of        approximately 100 nM. These lipid nanoparticles are capable of        delivering the nuclease to cells with high efficiency and low        toxicity;    -   b. A pharmaceutical formulation of the lipids, nuclease, and        exogenous donor DNA;    -   c. A pharmaceutical formulation of the lipids, nuclease and        exogenous donor DNA which is suitable for nebulization        (inhalation); and    -   d. A version of the invention which contains exogenous donor DNA        that when delivered with the TevCas9 nuclease in the lipid        nanoparticle is capable of integrating into the region between        or around the two sites targeted by the nuclease.

The novel chimeric nuclease compositions of the instant applicationcontain different combinations of an I-TevI domain, a linker domain, aCas9 domain and a guide RNA.

The versions that target the CFTR gene are comprised of:

-   -   i. An I-TevI domain of amino acid sequence according to SEQ ID        NO: 6;    -   ii. A linker domain according to any one of SEQ ID NOS: 7-12;    -   iii. A saCas9 domain of the amino acid sequence according to SEQ        ID NO: 13; and    -   iv. A guide RNA of the RNA sequence according to SEQ ID NO: 15        or 21.

The versions that target the EGFR gene are comprised of:

-   -   i. An I-TevI domain of amino acid sequence according to SEQ ID        NO: 6;    -   ii. A linker domain with any one of the amino acid sequences        according to SEQ ID NOS: 7-12;    -   iii. A saCas9 domain of the amino acid sequence according to SEQ        ID NO: 13; and    -   iv. A guide RNA of the RNA sequence in SEQ ID NO: 16.

The I-TevI domain of the preferred embodiment is a 93-amino acid I-TevIdomain of the Enterobacteria Phage T4 according to the followingsequence:

(SEQ ID NO: 6) MGKSGIYQIKNTLNNKVYVGSAKDFEKRWKRHFKDLEKGCHSSIKLQRSFNKHGNVFECSILEEIPYEKDLIIERENFWIKELNSKINGYNIA

The saCas9 of the preferred embodiment is a polypeptide comprised of1,053 amino acids according to the following sequence:

(SEQ ID NO: 13) MKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQHIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKG

The saCas9 with a Glu¹⁰ mutation of the preferred embodiment is apolypeptide comprised of 1,053 amino acids according to the followingsequence (the mutation is underlined):

(SEQ ID NO: 14) MKRNYILGLEIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQHIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKG

The guide RNA of the version that targets the CFTR gene is comprised of101 ribonucleotides according to the sequences:

(SEQ ID NO: 15) GCGUCAUCAAAGCAUGCCAACGUUUUAGUACUCUGGAAACAGAAUCUACUAAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGAU UUU (SEQ ID NO: 21)AUAUCAUUGGUGUUUCCUAUGGUUUUAGUACUCUGGAAACAGAAUCUACUAAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGAU UUU

The guide RNA of the version that targets the EGFR gene is 101ribonucleotides in length according to the following sequence:

(SEQ ID NO: 16) AAUUUUAACUUUCUCACCUUCGUUUUAGUACUCUGGAAACAGAAUCUACUAAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGAU UUU.

The linkers used in any of the above constructs may be selected from thegroup consisting of:

MUTATION(S) (indicated SEQUENCE (amino acid count) by underline)SEQ ID NO: DATFGDTCSTHPLKEEIIKKRSETFKAKMLKLGPDGRKALYSKPGSKN V117F  7GRWNPETHKFCKCGVRIQTSAYTCSKCRNGGSGGS (83 AA)DATFGDTCSTHPLKEEIIKKRSETVKAKMLKLGPDGRKALYSRPGSKS K135R  8GRWNPETHKFCKCGVRIQTSAYTCSKCRNGGSGGS (83 AA) N140SDATFGDTCSTHPLKEEIIKKRSETFKAKMLKLGPDGRKALYSRPGSKS V117F  9GRWNPETHKFCKCGVRIQTSAYTCSKCRNGGSGGS (83 AA) K135R N140SDATFGDTCSTHPLKEEIIKKRSETFKAKMLKLGPDGRKALYSRPGSKS K135R 10GRWNPETHKFCKCGVRIQTSAYTCSKCRGGSGGTGGS (86 AA) N140SDATFGDTCSTHPLKEEIIKKRSETFKAKMLKLGPDGRKALYSRPGSKS K135R 11GRWNPETHKFCKCGVRIQTSAYTCSKCRGGGGSGGGGS (87 AA) N140SDATFGDTCSTHPLKEEIIKKRSETFKAKMLKLGPDGRKALYSRPGSKS K135R 12GRWNPETHKFCKCGVRIQTSAYTCSKCRKESGSVSSEQLAQFRSLD N140S (95 AA)

Synthesis Example 1: A Method to Manufacture the TevCas9 Nuclease

The DNA coding sequences of the above mentioned I-TevI domain, linkerdomain and Cas9 domain are synthesized as one contiguous DNA sequenceusing techniques known in the art. Gene synthesis was conducted by BioBasic Inc. (Markham, On, Canada). Briefly, short oligonucleotides(˜50-60 base pairs) are synthesized which contain regions of overlap tocover the entire sequences of I-TevI domain, linker domain and Cas9domain. The oligonucleotides are mixed together in blocks ofapproximately 1 kilobase of the sequence to be synthesized andpolymerase chain reaction (PCR) is used to synthesize these ˜1 kilobaseblocks. The ˜1 kilobase blocks are then mixed and subjected to PCR tosynthesis the I-TevI domain, linker domain and Cas9 domain. Further, toenhance expression of TevCas9 in E. coli and simplify restriction enzymedigestion, the DNA sequence of TevCas9 was optimized prior to synthesis.First, three-base pair DNA codons that are infrequently used byEscherichia coli (“E. coli”) were replaced with those that occur morefrequently (for example, of the 6 codons coding for the amino acidarginine, the relative abundance of the codon AGG is 0.03 compared to0.42 for the codon CGT). In total, 37% of the codons were changed tothose preferred by E. coli. Second, the content on the nucleotidescytosine and guanine was increased from 39.6% to 48.6%. Third, two E.coli ribosome binding sites were removed from the sequence. Fourth, aNdeI restriction endonuclease site was removed from the internalsequence. The contiguous DNA is digested with the restrictionendonucleases NdeI and BamHI (New England Biolabs, Ipswich, Mass.,United States), whose target sites occur only once in the DNA sequence,and then inserted using DNA ligase (New England Biolabs, Ipswich, Mass.,United States) into a similarly digested pET-11a expression vector (EMDMillipore, Burlington, Mass., United States) suitable for expression ofTevCas9 in E. coli. The pET-11a vector containing TevCas9 is transformedinto the E. coli expression strain T7 Express (New England Biolabs#C2566, Ipswich, Mass., United States) which has been optimized forexpression of proteins, including nucleases. Alternatively, the E. coliexpression strain BL-21(DE3) (New England Biolabs #C2527, Ipswich,Mass., United States) is used. Successful transformations are confirmedby resistance of the E. coli to ampicillin or tetracycline and thecoding sequence of TevCas9 is verified by DNA sequencing of theexpression vector derived from the transformed E. coli. The transformedE. coli is grown at 37° C. to an optical density of 0.4 to 0.6 asmeasure by spectrophotometry at a wavelength of 600 nM and theexpression of the TevCas9 protein from the pET-11a vector in thetransformed E. coli expression strain is induced using IPTG for 10-12hours at 16° C. Successful expression of TevCas9 is verified by thepresence of an approximately 150 kDa band on a Coomassie-stainedSDS-polyacrylamide gel in a sample of the induced material compared tothe uninduced sample. The E. coli cells are harvested by centrifugationand resuspended in lysis buffer comprising 10 mM imidazole (Sigma, St.Louis, Mo., United States), 300-500 mM sodium chloride (Sigma-Aldrich,St. Louis, Mo., United States) and 50 mM sodium phosphate (dibasic)(Sigma, St. Louis, Mo., United States), pH 8.0 [Buffer 1].Alternatively, 10 mM Tris Hydrochloride (Sigma, St. Louis, Mo., UnitedStates), pH 8 is substituted for substituted for sodium phosphate(dibasic) in Buffer 1. The E. coli is lysed by homogenization using ahigh pressure liquid, homogenizer (Avestin Inc., Ottawa, ON, Canada)operated at 600-1000 bar, or any other suitable lysis method known inthe art, such as sonication using a sonifier (Branson Ultrasonics Corp,Danbury, Conn., United States) lysozyme treatment, homogenization usinga French pressure cell (Glen Mills Inc., Clifton, N.J., United States)or homogenization using a Dounce homogenizer (Corning Inc., Corning,N.Y., United States). The lysed material is centrifuged at 12,000 rpm at4° C. for 20-30 mins and the supernatant containing soluble TevCas9 isused for the subsequent purification steps. The pellet contains celldebris, insoluble intracellular material, as well as any insolubleTevCas9. Successful lysis and solubility is verified by the presence ofan approximately 150 kDa band on a Coomassie-stained SDS-polyacrylamidegel in a sample of the supernatant when compared to a resuspended sampleof the pellet.

The TevCas9 nuclease is purified in the following steps:

-   -   1. The lysate containing the nuclease is applied to an        immobilized metal affinity chromatography (IMAC) column (GE        Healthcare Bio-Sciences AB, Uppsala, Sweden) which binds the        nuclease.    -   2. The IMAC column is washed with Buffer 1.    -   3. The TevCas9 remaining bound to the column is eluted with a        solution comprising 250 mM imidazole (Sigma, St. Louis, Mo.,        United States), 300 mM-500 mM sodium chloride (Sigma-Aldrich,        St. Louis, Mo., United States) and 50 mM sodium phosphate        (dibasic) (Sigma, St. Louis, Mo., United States), pH 7.6-8.0        [Buffer 2]. Alternatively, 10 mM Tris Hydrochloride (Sigma, St.        Louis Mo., United States), pH 7.6-8 is substituted for        substituted for sodium phosphate (dibasic) in Buffer 2.    -   4. The eluate is treated with Tobacco Etch Virus (TEV) protease        (New England Biolabs, Ipswich, Mass., United States) and        incubated with the appropriate guide RNA. The guide RNA is        synthesized by Integrated DNA Technology Inc. (Coralville, Iowa,        United States).    -   5. The treated eluate is re-applied to the IMAC column and the        flow-through containing the TevCas9 nuclease and guide RNA is        collected.    -   6. Successful purification of the TevCas9 nuclease is confirmed        by the presence of a 150 kilodalton protein band on a        Coomassie-stained SDS-polyacrylamide gel. Successful        co-purification of TevCas9 with the guide RNA is confirmed by        treating a sample of the eluate with Proteinase K (New England        Biolabs, Ipswich, Mass., United States), then splitting the        sample in two and further treating one subsample with RNase A        (New England Biolabs, Ipswich, Mass., United States) and the        other in control buffer without RNase A. A ˜100 nucleotide RNA        band will be visible on an urea-polyacrylamide gel in the        control sample and will be absent in the RNase A-treated sample.    -   7. The solution containing the TevCas9 nuclease and guide RNA is        dialyzed into a solution comprising phosphate buffered saline,        pH 7.4.

Example 2: A Method to Manufacture the Lipid Nanoparticles

The lipid nanoparticles of the preferred embodiment are comprised of oneof the following mixtures:

-   -   I. Lipid nanoparticle No. 1 comprises DOPE (Avanti Polar Lipids,        Alabaster, Ala., United States) and MPEG-5000-DMPE (Avanti Polar        Lipids, Alabaster, Ala., United States) in a molar ratio of        2:0.05, respectively;    -   II. Lipid nanoparticle No. 2 comprises DPPC (Avanti Polar        Lipids, Alabaster, Ala., United States), cholesterol (SUPELCO,        Bellefonte, Pa., United States) and DOBA (Sigma, St. Louis, Mo.,        United States) in a molar ratio of 7:2:1, respectively; and    -   III. Lipid nanoparticle No. 3 comprised DPPC, cholesterol and        MPEG-5000-DMPE (Avanti Polar Lipids, Alabaster, Ala., United        States) in a molar ratio of 4:1:0.125, respectively.

Lipid nanoparticles are manufactured to a mean diameter of approximately100 nM.

One of the lipid mixtures No. 1-3 is selected. For example, DOPE andMPEG-5000-DMPE are mixed together in the appropriate molar ratios in anorganic solvent, such as chloroform. The organic solvent is thenevaporated and the dried lipid mixture is re-suspended using vigorousvortexing in a solution comprising phosphate buffered saline, pH 7.4.The re-suspended lipid mixture is then extruded through a 100 nMpolycarbonate membrane (T&T Scientific Corporation, Knoxville, Tenn.,United States) equilibrated in phosphate buffered saline to create lipidnanoparticles of an approximate mean diameter of 100 nM. The solution isfilter sterilized through 0.2 μM sterile filter (VWR Scientific, Radnor,Pa., United States). The mean diameter and size distribution of thelipid nanoparticles is determined by Dynamic Light Scattering (DLS)using a Zetasizer (Malvern Panalytical Ltd, Malvern, United Kingdom), oranother suitable technique, known in the art.

Example 3: Composition of the Donor DNA

The donor DNA comprises DNA sequences that are intended to repair agenetic defect. It also comprises DNA sequences which are not found inthe target genomic DNA; these sequences do not interfere with the normalgene function but are intended to knockout the I-TevI and or Cas9 sitesand/or introduce one or more DNA sequences which are used to track thesuccessful repair of the target gene. Examples of donor DNA include, butare not limited to the following:

-   -   I. Linear single-strand DNA of varying lengths comprising        homologous regions flanking the sites targeted/cleaved by        TevCas9;    -   II. Linear double-strand DNA of varying lengths comprising        homologous regions flanking the sites targeted/cleaved by        TevCas9;    -   III. Double-strand DNA of the same length cleaved by the        nuclease and also comprising complimentary DNA ends to those        cleaved by TevCas9;    -   IV. Circular double-strand DNA comprising homologous regions        flanking the sites targeted/cleaved by TevCas9; and    -   V. Circular double-strand DNA comprising an I-TevI target site        and Cas9 target site where the product cleaved from the        double-strand DNA contains complimentary ends to those cleaved        by TevCas9.

Example 4: A Method for Assembling the Lipid-Encapsulated TevCas9 andTransfecting Cells

For ex vivo cell transfections: To assemble the lipid-encapsulatedTevCas9, a lipid nanoparticle is mixed with the TevCas9 in a 2000:1molar ratio in Dulbecco's Modified Eagle's Medium (DMEM) (Sigma, St.Louis, Mo., United States) and incubated at room temperature for 10minutes. Cells are transfected with 8.7×10E-17 to 3.1×10E-17 moles oflipid-encapsulated TevCas9 per cell.

For in vivo cell transfection: To assemble the lipid-encapsulatedTevCas9, a lipid nanoparticle is mixed with the TevCas9 in a 2000:1molar ratio in phosphate buffered saline and incubated at roomtemperature for 10 minutes. The molar ratio of lipid-encapsulatedTevCas9 per cell for in vivo transfections is to be determined.

Other Embodiments

The nuclease might contain different combinations of the I-TevI domain,linker domain, Cas9 domain or guide RNA as highlighted below.

Modifications of the I-TevI Domain: Other versions of the I-TevInuclease domain might contain different combinations of mutations toalter the site targeted by the I-TevI domain or the activity of theI-TevI domain, including mutations that alter the sequence recognized byI-TevI, such as K26 and/or C39. Other versions of the nuclease mightsubstitute the I-TevI domain with other GIY-YIG nuclease domains, suchas I-BmoI, Eco29kI, etc. Other versions do not contain Met¹ as a resultof processing when expressed in E. coli.

Modifications of the Linker Domain: The linker domain might comprise onemore of the following to alter binding specificity or activity ofTevCas9, including: a). The I-TevI linker domain comprising one or moremutations to amino acid T95, V117, K135, Q158 or N140; b). The linkermight contain various combinations of the amino acids shown in SEQ IDNO: 9-12.

Modifications of the Cas9 Domain: Other versions of the Cas9 domainmight contain the following: a). A version of the saCas9 domaincomprising a D10E mutation (SEQ ID NO:14); b). A version of the saCas9domain that nicks target DNA on one strand of the target DNA, forexample the H557A mutation (SEQ ID NO: 17); c). A version of the saCas9domain that binds target DNA but does not cleave it, for examplemutations at both D10A and H557A mutations (SEQ ID NO: 18); d). Aversion of the previously described spCas9 EQR mutant comprising themutations D1135E, R1335Q and T1337R combined with the D10E mutation (SEQID NO: 19); and e). A version of the previously described spCas9 EQRmutant comprising the mutations D1135E, R1335Q and T1337R combined withthe D10E mutation and a mutation that nicks target DNA on one strand ofthe target DNA, for example the H840A mutation (SEQ ID NO: 20). Otherversions of the saCas9 domain do not contain Met¹.

Other versions might substitute other nucleases or DNA binding domainsfor the Cas9 domain, such as: a). Meganucleases such as the familiesLAGLIDADG, His-Cys Box, H—N—H, PD-(D/E)xK, Vsr-like, etc.; b).Zinc-finger nucleases; c). Other CRISPR proteins such as scCas9, fnCas9,cjCas9, Cpf1, Cas12a, Cas13a, Cas3, etc.; and d). Other DNA bindingdomains such as zinc-finger motifs, TALE activator domains, etc.

Modifications of the Guide RNA: a). Other versions of the guide RNAmight target the same region of DNA in the CFTR gene or EGFR gene, butcontain different sequences to account for genetic polymorphism inpopulations; b). Other versions of the guide RNA might target differentsequences in the CFTR gene or EGFR gene; c). Other version of the guideRNA might target other sequences in a genome to retarget the nuclease toadditional clinically relevant targets; d). Other versions of the guideRNA might contain bridged nucleic acids (“BNAs”) to enhance target sitespecificity; and e). Other versions might contain a mixture of guideRNAs to target multiple sequences within the same gene.

Modifications of the Lipid Nanoparticles: a). Other versions of thelipid nanoparticle No. 1, 2 or 3 might have different ratios of eachlipid component; b). Other versions of the lipid nanoparticle might havedifferent mean diameters; c). Other versions of the lipid nanoparticlemight include different cationic or neutral lipids; d). Other versionsof the lipid nanoparticle might include peptides that target specificcell types; e). Other versions of the lipid nanoparticle might includecompounds that bind DNA, such as GL67 (N⁴—Cholesteryl-Spermine); f). thelipid nanoparticle might be lyophilized for enhanced stability; and g).the lipid nanoparticle might be resuspended in a solution other thanphosphate buffered saline, such as sterile isotonic saline, water forinjection, etc.

Modifications of the Composition of the donor DNA: a). Other version ofthe linear double-strand donor DNA might contain longer regions ofsingle-strand DNA that is complementary to the target sequence; and b).Other versions of the circular double-strand DNA might contain other DNAsequences intended to increase the rate of homology-directed repair.

Variations to the method of assembling the lipid-encapsulated nucleaseand transfecting cells: a). Other versions of the lipid-encapsulatednucleases might contain different molar ratios of lipid nanoparticle tonuclease; b). Media other than DMEM or phosphate buffered saline mightbe used for the incubation step; c). The nuclease and lipidnanoparticles might be incubated for less or more than 10 minutes; andd). Other molar amounts of nuclease per cell might be used in atransfection reaction.

Variations to the method to manufacture the nuclease: a). Other E. coliexpression strains might be used, such as LS5218 (Escherichia coliGenetic Stock Center—Yale University, New Haven, Conn., United States)or BL21-DE3 (New England Biolabs, Ipswich, Mass., United States) b).Buffer 1 or 2 might contain different concentrations of imidazole,sodium chloride, sodium phosphate (dibasic), or tris hydrochloride andbe buffered to a different pH; c). Other processing steps might be used,such as cation or anion exchange chromatography; d). The nuclease mightbe dialyzed into a solution other than phosphate buffered saline, suchas sterile isotonic saline, water for injection, etc.; e). the nucleasemight be lyophilized for enhanced stability; f). the guide RNA may beco-expressed from the pACYC-Duet1 expression vector (EMD Millipore,Burlington, Mass., United States). The DNA coding sequence of the guideRNA is synthesized (Integrated DNA Technology Inc., Coralville, Iowa,United States), digested with restrictions endonucleases and insertedinto similarly-digested second expression site in the pACYC-Duet1expression vector; and g). The guide RNA may be synthesized fromdouble-strand DNA by transcribing the guide using the T7 RNA PolymeraseHiScribe Kit (New England Biolabs #E2040S, Ipswich, Mass., UnitedStates) and purifying the guide using an RNA Cleanup Kit (New EnglandBiolabs #T2030L, Ipswich, Mass., United States).

Testing Example 1: A Method to Demonstrate Correction of CFTR Delta F508and CFTR Protein Functionality in a Model Cell Line

A culture of immortalized epithelial cells homozygous for the CFTR deltaF508 mutation, such as the CuFi-1 cell line (ATCC® CRL-4013™, AmericanType Culture Collection, Manassas, Va., United States), is treated witha range of concentrations of lipid-encapsulated TevCas9 and donor DNA(Specific Biologics, Toronto, ON, Canada) in a pharmaceuticalformulation targeted to the CFTR delta F508 mutation. An appropriatecontrol cell line, such as NuLi-1 (ATCC® CRL-4011™, American TypeCulture Collection, Manassas, Va., United States) immortalizedepithelial cells homozygous for wild-type CFTR is also be used.

The proportion of cells with CFTR delta F508 corrected relative touncorrected cells is measured by the T7 endonuclease I assay (EnGen®Mutation Detection Kit, New England Biolabs #E3321, Ipswich, Mass.,United States), restriction endonuclease digestion (New England Biolabs,Ipswich, Mass., United States) of PCR-amplified target site, deep genesequencing using an Illumina MiSeq system and barcoded primers flankingthe target site (Illumina, San Diego, Calif., United States), or othersuitable method. The effects of TevCas9 treatment in the control cellline (e.g. NuLi-1 (ATCC® CRL-4011™, American Type Culture Collection,Manassas, Va., United States) is measured. CFTR functionality ismeasured using short circuit current measurements in an Ussing Camber(Warner Instruments, Hamden, Conn., United States) in the presence of achloride ion gradient in the treated CuFi-1 culture (ATCC® CRL-4013™American Type Culture Collection, Manassas, Va., United States) versusmock-treated CuFi-1 culture (ATCC® CRL-4013™, American Type CultureCollection, Manassas, Va., United States). The effects of TevCas9treatment in the control cell line (e.g. NuLi-1) is also measured.

To demonstrate disruption of the EGFR exon 19 deletion mutations(s) andEGFR expression and activity in a model cell line, a culture ofimmortalized epithelial cells expressing an EGFR exon 19 deletionmutation(s), such as the HCC827 cell line (ATCC® CRL-2868™ American TypeCulture Collection, Manassas, Va., United States) is treated with arange of concentrations of lipid-encapsulated TevCas9 (SpecificBiologics, Toronto, ON, Canada) in a pharmaceutical formulation ofphosphate buffered saline, sterile isotonic saline or water forinjection targeted to the EGFR exon 19 deletion. Appropriate controlcell lines, such as NuLi-1 (ATCC® CRL-4011™, American Type CultureCollection, Manassas, Va.) or immortalized epithelial cell lineshomozygous for wild-type EGFR, are used.

The proportion of cells with the EGFR exon 19 deletion disruptedrelative to uncorrected cells is measured by the T7 endonuclease I assay(EnGen® Mutation Detection Kit, New England Biolabs #E3321, Ipswich,Mass., United States), restriction endonuclease digestion (New EnglandBiolabs, Ipswich, Mass., United States) of PCR-amplified target site,deep gene sequencing using an Illumina MiSeq system and barcoded primersflanking the target site (Illumina, San Diego, Calif., United States),or other suitable method. The effects of TevCas9 treatment in thecontrol cell line (e.g. NuLi-1 (ATCC® CRL-4011™, American Type CultureCollection, Manassas, Va., United States)) is also measured. EGFRprotein expression and activity are measured using an enzyme-linkedimmunosorbent assay (ELISA) (Sigma, St. Louis, Mo., United States) thatdetects phosphorylated (i.e. activated), unphosphorylated and total EGFRprotein in the treated HCC827 culture (ATCC® CRL-2868™, American TypeCulture Collection, Manassas, Va., United States) versus mock-treatedHCC827 culture. The effects of TevCas9 treatment in the control cellline (e.g. NuLi-1 (ATCC® CRL-4011™, American Type Culture Collection,Manassas, Va., United States)) is also measured.

Example 1: Animal Model Testing Planned to Show Efficacy and DetermineDose-Limiting Toxicity

In an example method to demonstrate correction of CFTR delta F508 and/orCystic Fibrosis symptoms with lipid-encapsulated TevCas9 treatment in ananimal model (for example, mouse, rat, minipig or ferret), thelipid-encapsulated TevCas9 in a pharmaceutical formulation of phosphatebuffered saline, sterile isotonic saline or water for injection targetedto the CFTR delta F508 is delivered directly to the lungs by eitherintubation or intranasal delivery. The procedure time is approximately30-6000 seconds per treatment, depending on the animal model used.

In another method, the lipid-encapsulated TevCas9 in a pharmaceuticalformulation targeted to the CFTR delta F508 is nebulized with acommercial nebulizer (Aeroneb®, AeroEclipse®, (Trudell Medical, London,ON, Canada)) or PARI-LC Plus®, (PARI USA, Midlothian, Va., UnitedStates)). The average size of the lipid nanoparticle of approximately100 nM is confirmed post-nebulization by Dynamic Light Scattering (DLS)using a Zetasizer (Malvern Panalytical Ltd, Malvern, United Kingdom), oranother suitable technique, known in the art. The composition andconcentration of the lipid-encapsulated TevCas9 is confirmedpost-nebulization using the MicroGram Lipid Assay Kit (ProFoldin,Hudson, Mass., United States) and the presence of an approximately 150kDa band on a Coomassie-stained SDS-polyacrylamide gel. For measurementof the rate of gene correction, a representative ovine (minipig) animalmodel (Exemplar Genetics, Sioux City, Iowa, United States) that ishomozygous for the CFTR delta F508 mutation is exposed through themouth, nose or directly to the lungs with the lipid-encapsulated TevCas9targeted to CFTR delta F508, as well as a suitable control. Generalmaintenance of these animals includes breeding and farrowing;age-appropriate, bio-secure housing; sound nutrition; basic vaccinationsand veterinary care; and documentation consistent with animal welfareguidelines. Maintenance of these animals specific to the CFTR delta F508may include one of more of the following: surgery to address intestinalobstruction; pancreatic enzyme replacement therapy; vitamins and H2blockers; and/or proton pump inhibitors to improve gastric acid control.The minipigs are treated with a range of concentrations oflipid-encapsulated TevCas9 that are predicted to be effective from themodel cell line studies above for 2 days to 4 weeks for acute toxicitystudies and up to 24 months for chronic toxicity studies.

The general health of the animal is monitored post-treatment to assessfor any treatment-related adverse events, such as changes in behavior,weight, or food consumption; immune responses; changes to cardiovascularhealth; mortality, etc. Other efficacy measures post-treatment mayinclude:

-   -   I. Forced-expiration, such as forced expiratory volume (or other        suitable method) in each animal post-treatment;    -   II. Overall survival of each animal relative to the control;    -   III. Other measures of lung function (for example, utilization        of mechanical ventilator that can perform general lung function        assessments); and    -   IV. Measurements of the mutation in vivo through tissue sampling        and mutation detection methods, such as by polymerase chain        reaction.        After the treatment(s) with lipid-encapsulated TevCas9, the        animals are sacrificed and the lung and tracheal tissue are        harvested.

The proportion of cells with CFTR delta F508 corrected relative touncorrected cells is measured by the T7 endonuclease I assay (EnGen®Mutation Detection Kit, New England Biolabs #E3321, Ipswich, Mass.,United States), restriction endonuclease digestion (New England Biolabs,Ipswich, Mass., United States) of PCR-amplified target site, deep genesequencing using an Illumina MiSeq system and barcoded primers flankingthe target site (Illumina, San Diego, Calif., United States) or othersuitable method.

In a method to demonstrate disruption of the EGFR exon 19 deletionmutation(s) and/or Non-small-cell lung cancer (NSCLC) symptoms withTevCas9 treatment in an animal model, the lipid-encapsulated TevCas9targeted to the EGFR exon 19 deletion mutations in a pharmaceuticalformulation of phosphate buffered saline, sterile isotonic saline orwater for injection is delivered directly to the lungs through themouth, nose or directly to the lungs. The procedure time isapproximately 30-6000 seconds per treatment, depending on the animalmodel used.

In another method, the lipid-encapsulated TevCas9 targeted to the EGFRexon 19 deletion mutations in a pharmaceutical formulation is nebulizedwith a commercial nebulizer ((Aeroneb®, AeroEclipse®, (Trudell Medical,London, ON, Canada) or PARI-LC Plus®, (PARI USA, Midlothian, Va., UnitedStates)). The average size of the lipid nanoparticle of approximately100 nM is confirmed post-nebulization by Dynamic Light Scattering (DLS)using a Zetasizer (Malvern Panalytical Ltd, Malvern, United Kingdom), oranother suitable technique, known in the art. The composition andconcentration of the lipid-encapsulated TevCas9 nanoparticle isconfirmed post-nebulization using the MicroGram Lipid Assay Kit(ProFoldin, Hudson, Mass., United States) and the presence of anapproximately 150 kDa band on a Coomassie-stained SDS-polyacrylamidegel. For measurement of the rate of gene disruption, a representativemurine (mouse) animal model that is homozygous for an EGFR exon 19deletion mutation(s) is exposed through the nose, mouth or directly tothe lungs with the lipid-encapsulated TevCas9 targeted to EGFR exon 19deletion. The mice are treated with a range of concentrations of TevCas9that are predicted to be effective from the model cell line studies for2 days to 4 weeks for acute toxicity studies and up to 24 months forchronic toxicity studies.

The general health of the animal is monitored post-treatment to assessfor any treatment-related adverse events, such as changes in behavior,weight, or food consumption; immune responses; changes to cardiovascularhealth; mortality, etc. Other efficacy measures post-treatment mayinclude:

-   -   I. Quantification of EGFR-activating protein through positron        emission tomography (PET) with an EGFR mutant tracer;    -   II. Overall survival of each animal relative to the control(s);        and    -   III. Measures of tumor formation/reduction in each animal over        time.        Measurements of the mutation in vivo through tissue sampling and        mutation detection methods, such as the Cobas® EGFR mutation        test version 2 (Roche Diagnostics, Risch-Rotkreuz, Switzerland).        After treatment with nebulized lipid-encapsulated TevCas9, the        animals are sacrificed and the lung and tracheal tissue are        harvested.

The proportion of cells with the EGFR exon 19 deletion mutationdisrupted relative to undisrupted is measured by the T7 endonuclease Iassay (EnGen® Mutation Detection Kit, New England Biolabs #E3321,Ipswich, Mass., United States), restriction endonuclease digestion (NewEngland Biolabs, Ipswich, Mass., United States) of PCR-amplified targetsite, deep gene sequencing using an Illumina MiSeq system and barcodedprimers flanking the target site (Illumina, San Diego, Calif., UnitedStates) or other suitable method. EGFR protein expression and activityin cells of the harvest tissues are measured using an enzyme-linkedimmunosorbent assay (ELISA) (Sigma, St. Louis, Mo., United States) thatdetects phosphorylated (i.e. activated), unphosphorylated and total EGFRprotein. Determination of dose-limiting toxicity to enablefirst-in-human clinical studies is based on the predicted effectivedose(s) from the animal model studies discussed above, a range ofconcentrations (in milligrams per kilogram body weight, for example) oflipid-encapsulated TevCas9 is nebulized and delivered to an appropriateanimal model for toxicology studies, such as the cynomolgus monkey orother non-human primate. The general health of the animals is monitoredfor any treatment-related adverse events, such as changes in behavior,weight, or food consumption; immune responses; changes to cardiovascularhealth; mortality, etc. Other measures of efficacy may be measured inthe studies, including those described above.

Therapeutic Effect

The novel chimeric nucleases of the instant invention have beenintentionally designed to modify the DNA of lung epithelial cells totreat monogenetic diseases although they are capable of working in othercell types or in the cells of other organisms such as bacteria, yeast,insect, plant or other mammals in vivo or ex vivo to treat monogeneticor polygenetic and infectious diseases.

Example 1: A Method of Targeted Insertion or Replacement of all or aPortion of a DNA Sequence in the Genome of Human Cells

FIG. 2A to 2E illustrate the mechanism of action of cellular uptake ofthe novel chimeric nuclease of the instant invention. As illustrated inFIG. 2A, a cell 20 or cells 20 are exposed to the novellipid-encapsulated nuclease particles 21 containing the TevCas9 25either by in vivo or ex vivo administration. As shown in FIG. 2B, thelipid-encapsulated nuclease particle 21 is endocytosed into the cell 20.The endosome 22 goes through a maturation process in the cytosol and istargeted for degradation (FIG. 2C). On certain occasions, the TevCas9 25can escape the endosome 22 and enter the cytosol (FIG. 2D). Ineukaryotic organisms, the nuclease (TevCas9) 25 is targeted to thenucleus 23 of the cell 20 through one or more nuclear-localizationsequences (“NLS”). As depicted in FIG. 2E, through its nuclearlocalization sequence, TevCas9 25 can enter the nucleus 23 and when inthe nucleus 23, the TevCas9 nuclease 25 binds to and cleaves 26 thetarget genomic DNA 24 sequence.

FIG. 3A to 3E illustrate the mechanism of the TevCas9 nuclease incutting DNA. FIG. 3A is representation of the key features of TevCas9bound to its target genomic DNA sequence 24 is shown prior to thecleavage reaction. The I-TevI domain 27 targets the I-TevI TargetSequence 29. The linker domain 30 joins the I-TevI domain 27 with theCas9 domain 28 which targets the Cas9 Target Sequence 31. The genemutation 32 is surrounded by or in close proximity to the I-TevI TargetSequence 29 and the Cas9 Target Sequence 31. As shown in FIG. 3B, theTevCas9 25 cleaves the target sequence leaving a deletion product 34 ofa predictable size with non-complementary DNA ends 35, 36. FIG. 3Cillustrates that in the presence of single-stranded donor DNA withhomology arms 37, the cell 20 can insert the donor DNA 37 sequence nearthe cut sites through the homology-directed repair (HDR) pathway 38.FIG. 3D illustrates that in the presence of donor DNA 39 with compatibleDNA ends to those cleaved by TevCas9 25, the cell 20 can insert thedonor DNA sequence 39 between the cut sites through directed-ligationusing the non-homologous end joining (NHEJ) pathway 40. In the absenceof donor DNA, the cell 20 can join the DNA ends through the NHEJ pathway40 (FIG. 3E).

Example 2: The Treatment of Cystic Fibrosis

For the treatment of cystic fibrosis, the exogenous donor DNA contains aDNA sequence, which repairs the CFTR delta F508 mutation involving amethod of targeted deletion of a defined length of a DNA sequence inhuman somatic cells to stimulate homology-directed repair usingexogenous donor DNA as a template (FIG. 3C).

Example 3: The Treatment of Non-Small Cell Lung Cancer

For the application of treating non-small cell lung cancer, a version ofthe Cas9 domain which cuts only one strand of DNA (D10A or H557Amutation) or a nuclease deficient version (the D10A+H557A mutations) isused and the sequences targeted are EGFR exon 19 deletion mutations (SEQID NOS: 2-4). In this application, however, the nuclease does notcontain exogenous donor DNA. In the absence of exogenous donor DNA, thecell can remove the DNA sequence between the two sites targeted by thenuclease by non-homologous end joining (FIG. 3E).

Alternatively, the inhalation route is a fast and effective way ofdelivering medication locally to the lungs and for the systemicadministration of certain agents. Inhalation drug therapy is usedextensively to treat respiratory conditions such as asthma and ChronicObstructive Pulmonary Disease (COPD). Research is ongoing to developinhalation systems to treat cystic fibrosis.

The examples which follow are intended in no way to limit the scope ofthe disclosure but are provided to illustrate how to prepare and usecompounds disclosed herein. Many other embodiments of this disclosurewill be apparent to one skilled in the art.

A nebulizer is a device that delivers medication to the lungs in theform of an aerosolized vapor. Nebulizers are commonly used to treatrespiratory diseases such as asthma and COPD, for example, thenebulization of corticosteroids, although nebulization has also beenused for the treatment and prevention of lung infections, such asARIKAYCE® (Insmed Incorporated, Bridgewater, N.J., United States).

The nebulizer may require some procedure to prepare the liquid fornebulization. The medication is commonly held in liquid form in a cupinside the nebulizer chamber. Once loaded, the device is switched onwhich generates compressed air to convert the liquid into a vapor in thenebulization chamber. The patient puts the mouthpiece of thenebulization chamber into their mouth and takes a sharp, deepinhalation, holding their breath for 5-10 seconds to ensure themedication reaches the lower parts of the lung. There are a variety ofsuch devices. Many modern nebulizers are breath-actuated and rely on theforce of patient inhalation to entrain the aerosolized liquid from thedevice and thus ensure the medication is delivered only to the patientand not to the surrounding environment. This also ensures consistency ofdelivering a full dose of the medication to the patient.

The use of nebulizers is well known and nebulizers are commerciallyavailable from several sources, such as Aeroneb®, AeroEclipse®, (TrudellMedical, London, ON, Canada) or PARI-LC Plus®, (PARI USA, Midlothian,Va., United States). In an example of the present invention, a nebulizeris utilized for delivery of the lipid-encapsulated novel chimericnuclease comprising a modified I-TevI nuclease domain, a linker, and amodified RNA-guided nuclease Staphylococcus aureus Cas9 of the instantapplication to the lung epithelial tissue. A sterile liquid version ofthe therapeutic of interest is loaded into the nebulization chamber andis subsequently aerosolized and is inhaled by the patient into the lungvia deep breaths.

Some of the advantages of using a nebulizer versus oral or intravenousadministration are: less drug could be required compared to oral orintravenous administration, onset of action can be more rapid viainhalation compared to the oral route, adverse effects are potentiallyless severe due to local delivery of the medication to the lung tissuewhere the disease manifests itself, inhaled drug therapy is painless andrelatively comfortable for the patient which encourages compliance.

No non-invasive route of delivery provides the speed of action that aninhaled drug can provide. One of the advantages of inhaled drugs is thatthey are more rapidly absorbed than subcutaneously injected moleculesand provides a more immediate physiological response. Small or largemolecules, particularly hydrophobic molecules, can be absorbed withinseconds after inhalation and can thus be used to treat a wide variety ofsymptoms that come on suddenly or need long term administration. Pain,panic, anxiety, nausea, cardiovascular crises, bronchoconstriction,sleep induction, spasms, Parkinson's lock-up, and hot flashes are someof the rapid-onset conditions that are addressable with inhaledmedicines.

Most protein-based drug products have some water solubility and arerapidly and efficiently absorbed from the lungs. Those that are morehydrophobic are absorbed even more rapidly within seconds to a fewminutes. Those that are more hydrophilic are absorbed within minutes totens of minutes. In one example of the present invention, one vial isaseptically filled with a therapeutic dose of the lipid nanoparticlewhich is hydrophobic and another vial is aseptically filled with atherapeutic dose of a chimeric nuclease comprising a modified I-TevInuclease domain, a linker and a modified RNA-guided nucleaseStaphylacoccus aureus Cas9 which is water soluble and hydrophilic, fordelivery to the lungs of a therapeutic dose. The dose can range from 1to 1000 milligrams of each of the lipid nanoparticle and chimericnuclease with about 5 to 200 milligrams being preferred. The claimedlipid-encapsulated chimeric nuclease comprising a modified I-TevInuclease domain, a linker, and a modified RNA-guided nucleaseStaphylacoccus aureus Cas9 can be absorbed in the cells of the lungswithin a few hours and complete cleavage on DNA substrates in vitro hasbeen observed within 2 hours. Other nebulized therapies have beendelivered daily. Nebulized administration of the lipid-encapsulatedchimeric nuclease, therefore, can be daily or more infrequentlydepending on its efficacy on a per patient basis. The chimeric nucleasesare manufactured by BioVectra Corporation (Charlottetown, PE, Canada)and the vials are aseptically filled by Dalton Pharma Services(Mississauga, ON, Canada). The lipid nanoparticles are manufactured andthe vials are aseptically filled by Transferra Nanosciences Inc.(Burnaby, BC, Canada).

The dosage of any disclosed compositions will vary depending on thesymptoms, age and body weight of the patient, the nature and severity ofthe disorder to be treated or prevented, the route of administration,and the form of the subject composition. Any of the subject formulationsmay be administered in a single dose or in divided doses. Dosages forthe compositions may be readily determined by techniques known to thoseof skill in the art or as taught herein.

In certain embodiments, the dosage of the subject compounds willgenerally be in the range of about 1 to 1000 milligrams depending on thebody weight of the patient, specifically in the range of about 5 to 200milligrams.

An effective dose or amount, and any possible effects on the timing ofadministration of the formulation, may need to be identified for anyparticular composition of the disclosure. This may be accomplished byroutine experiment as described herein, using one or more groups ofanimals (preferably at least 5 animals per group), or in human trials ifappropriate. The effectiveness of any subject composition and method oftreatment or prevention may be assessed by administering the compositionand assessing the effect of the administration by measuring one or moreapplicable indices and comparing the post-treatment values of theseindices to the values of the same indices prior to treatment.

The precise time of administration and amount of any particular subjectcomposition that will yield the most effective treatment in a givenpatient will depend upon the activity, pharmacokinetics, andbioavailability of a subject composition, physiological condition of thepatient (including age, sex, disease type and stage, general physicalcondition, responsiveness to a given dosage and type of medication),route of administration, and the like. The guidelines presented hereinmay be used to optimize the treatment, e.g., determining the optimumtime and/or amount of administration, which will require no more thanroutine experimentation consisting of monitoring the subject andadjusting the dosage and/or timing.

While the subject is being treated, the health of the patient may bemonitored by measuring one or more of the relevant indices atpredetermined times during the treatment period. Treatment, includingcomposition, amounts, times of administration and formulation, may beoptimized according to the results of such monitoring. The patient maybe periodically re-evaluated to determine the extent of improvement bymeasuring the same parameters. Adjustments to the amount(s) of subjectcomposition administered and possibly to the time of administration maybe made based on these re-evaluations.

Treatment may be initiated with smaller dosages which are less than theoptimum dose of the compound. Thereafter, the dosage may be increased bysmall increments until the optimum therapeutic effect is attained.

The use of the subject compositions may reduce the required dosage forany individual agent contained in the compositions because the onset andduration of effect of the different agents may be complimentary.

Therapeutic efficacy of subject compositions may be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ and the ED₅₀.

The data obtained from the cell culture assays and animal studies may beused in formulating a range of dosage for use in humans. The dosage ofany subject composition lies preferably within a range of concentrationsthat include the ED₅₀ with little or no toxicity. The dosage may varywithin this range depending upon the dosage form employed and the routeof administration utilized. For compositions of the disclosure, thetherapeutically effective dose may be estimated initially from cellculture assays.

Formulations

Pharmaceutical compositions of the disclosure may be administered byvarious means, depending on their intended use, as is well known in theart. For example, compositions of the disclosure are to be administeredthrough nebulization. Alternatively, formulations disclosed herein maybe administered intravenously, subcutaneously, or intramuscularly. Theseformulations may be prepared by conventional means, and, if desired, thecompositions may be mixed with any conventional additive, such as anexcipient, a solubilizing agent, a suspension aid, an emulsifying agent,or preservative agent. The disclosed excipients may serve more than onefunction. For example, a solubilizing agent may also be a suspensionaid, an emulsifier, a preservative, and the like.

The formulations may conveniently be presented in unit dosage form andmay be prepared by any methods well known in the art of pharmacy. Theamount of a composition that may be combined with a carrier material toproduce a single dose vary depending upon the subject being treated, andthe particular mode of administration.

Methods of preparing these formulations include the step of bringinginto association compositions of the disclosure with the carrier and,optionally, one or more accessory ingredients. In general, theformulations are prepared by uniformly and intimately bringing intoassociation agents with liquid carriers.

It will be appreciated that a disclosed composition may includelyophilized or freeze-dried compounds disclosed herein. For example,disclosed herein are compositions that disclosed compounds crystallineand/or amorphous powder forms. Such forms may be reconstituted for useas e.g., an aqueous composition.

Liquid dosage forms for injection include pharmaceutically acceptablesolutions, emulsions, microemulsions, solutions and suspensions. Inaddition to the subject composition, the liquid dosage forms may containinert diluents commonly used in the art, such as, for example, water orother solvents, solubilizing agents and emulsifiers, such as ethylalcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol,glycerol, tetrahydrofuryl alcohol, and fatty acid esters of sorbitan,cyclodextrins, albumin, hyaluronic acid, chitosan and mixtures thereof.Polyethylene glycol (PEG) may be used to obtain desirable properties ofsolubility, stability, half-life, and other pharmaceuticallyadvantageous properties. Representative examples of stabilizingcomponents include polysorbate 80, L-arginine, polyvinylpyrrolidone,trehalose, and combinations thereof. Other excipients that may beemployed, such as solution binders or anti-oxidants include, but are notlimited to, butylated hydroxytoluene (BHT), calcium carbonate, calciumphosphate (dibasic), calcium stearate, croscarmellose, crosslinkedpolyvinyl pyrrolidone, citric acid, crospovidone, cysteine,ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, mannitol,methionine, methylcellulose, methyl paraben, microcrystalline cellulose,polyvinyl pyrrolidone, povidone, pregelatinized starch, propyl paraben,retinyl palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch(corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A,vitamin E (alpha-tocopherol), vitamin C and xylitol.

Ordinarily, an aqueous aerosol is made by formulating an aqueoussolution or suspension of a subject composition together withconventional pharmaceutically acceptable carriers and stabilizers. Thecarriers and stabilizers vary with the requirements of the particularsubject composition, but typically include non-ionic surfactants(Tweens, pluronics, or polyethylene glycol), innocuous proteins likeserum albumin, sorbitan esters, oleic acid, lecithin, amino acids suchas glycine, buffers, salts, sugars or sugar alcohols. Aerosols generallyare prepared from isotonic solutions.

It should be noted that excipients given as examples may have more thanone function. For example, a solubilizing agent may also be a suspensionaid, an emulsifier, a preservative and the like.

Examples of suitable aqueous and non-aqueous carriers which may beemployed in the pharmaceutical compositions of the disclosure includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate and cyclodextrins. Proper fluidity may be maintained,for example, by the use of coating materials, such as lecithin, by themaintenance of the required particle size in the case of dispersions,and by the use of surfactants.

The compositions of the present invention typically are ready toadminister, aqueous solutions which are sterile, storage-stable, andpharmaceutically acceptable without the need for reconstitution prior toadministration. The compositions of the present invention are suitablefor administration to a subject which means that they arepharmaceutically acceptable, non-toxic, do not contain any componentswhich would adversely affect the biological effects of the chimericnuclease, and have the pH close to that of the physiological conditionwhich avoids inhalation and/or injection site reactions. Thecompositions of the present invention do not, for example, comprise anycells.

The compositions are typically stored in a sealed container, vial orcartridge which is typically suitable for long term storage. “Suitablefor long-term storage” means that the vial, container or cartridge doesnot allow for the escape of components of the compositions of thepresent invention or the ingress of external components, such as,microorganisms when kept for at least 3 months at 25° C.

The compositions of the present invention are preferably administered bynebulization, typically breath-actuated nebulization.

The compositions of the present invention can also be administered byinjection as described herein.

The compositions of the present invention may be administered alone orin combination with an additional therapeutic agent, such as ananti-viral, an anti-microbial, a chemotherapeutic and an immunotherapy.

Vials, as used herein, can also comprise two containers one of whichcontains the chimeric nuclease or lipid particle, as described herein,in a lyophilized powder, as described below, and the second containercontains a liquid for reconstitution of the lyophilized powder. Thecontents of the two containers can be mixed prior to administration.

As discussed above the compositions of the present invention can beadministered by nebulization. Suitable volumes of the compositions ofthe present invention for nebulization include about 0.5 to about 1 ml,about 1 to about 2 ml, about 2 to about 10 ml, or about 10 to about 20ml.

In the compositions of the present invention the concentration of thechimeric nuclease is from about 0.1 mg/ml to about 10.0 mg/ml, fromabout 10.0 mg/ml to about 100.0 mg/ml, from about 30.0 mg/ml to about300.0 mg/ml, from about 500 mg/ml to about 2000 mg/ml and about 2.0mg/ml.

In the compositions of the present invention the concentration of thelipid nanoparticle is from about 0.1 mg/ml to about 10.0 mg/ml, fromabout 10.0 mg/ml to about 100.0 mg/ml, from about 30.0 mg/ml to about300.0 mg/ml, from about 500 mg/ml to about 2000 mg/ml and about 2.0mg/ml.

We claim:
 1. A nuclease comprising a modified Staphylococcus aureusCas9, wherein said modified Staphylococcus aureus Cas9 comprises anaspartic acid to glutamic acid substitution at an amino acidcorresponding to position 10 of SEQ ID NO.
 13. 2. The nuclease of claim1, wherein the modified Staphylococcus aureus Cas9 comprises thesequence of SEQ ID NO:
 14. 3. The nuclease of claim 1, wherein themodified Staphylococcus aureus Cas9 further comprises a histidine toalanine substitution at an amino acid corresponding to position 557 ofSEQ ID NO:
 13. 4. The nuclease of claim 1, further comprising an I-Tevlnuclease domain.
 5. The nuclease of claim 4, wherein the I-Tevl nucleasedomain comprises the amino acid sequence set forth in SEQ ID NO:
 6. 6.The nuclease of claim 5, wherein the I-Tevl nuclease domain comprises asubstitution or deletion of the methionine at position 1 of SEQ ID NO:6.
 7. The nuclease of claim 1, further comprising a linker coupling themodified Staphylococcus aureus Cas9 and the I-Tevl nuclease domain. 8.The nuclease of claim 7, wherein the linker comprises an amino acidsequence set forth in any one or more of SEQ ID NO: 7 to SEQ ID NO 12.9. The nuclease of claim 1, bound to a guide RNA.
 10. The nuclease ofclaim 9, wherein the guide RNA comprises one or more bridged nucleicacids.
 11. A chimeric nuclease comprising an I-Tevl nuclease domain, alinker, a Staphylococcus aureus Cas9, and a guide RNA.
 12. The chimericnuclease of claim 11, wherein the Staphylococcus aureus Cas9 comprisesan aspartic acid to glutamic acid substitution at an amino acidcorresponding to position 10 of SEQ ID NO
 13. 13. The chimeric nucleaseof claim 11, wherein the Staphylococcus aureus Cas9 comprises thesequence of SEQ ID NO:
 14. 14. The chimeric nuclease of claim 11,wherein the I-Tevl nuclease domain comprises the amino acid sequence setforth in SEQ ID NO:
 6. 15. The chimeric nuclease of claim 14, whereinthe I-Tevl nuclease domain comprises a substitution or deletion of themethionine at position 1 of SEQ ID NO.
 6. 16. The chimeric nuclease ofclaim 11, wherein the linker comprises an amino acid sequence set forthin any one or more of SEQ ID NO: 7 to SEQ ID NO
 12. 17. A formulationcomprising the chimeric nuclease of claim 11 and a lipid nanoparticle.18. The chimeric nuclease of claim 11 for use in a method to geneticallymodify the genome of a cell or organism.
 19. A nucleic acid encoding thenuclease of claim
 1. 20. A nucleic acid encoding the chimeric nucleaseof claim 11.